PROJECT SUMMARY AND ABSTRACT Coinhibitory immunoreceptor programmed cell death-1 (PD-1, CD279) plays a significant role in maintaining peripheral tolerance and protecting healthy tissues from inflammatory tissue damage. Tumors have exploited the PD-1 pathway to evade immune-mediated destruction. Although PD-1 pathway inhibitors have revolutionized cancer care, only a subset of patients respond and show long-lasting remission. Despite this clinical success, the intracellular signaling mechanisms underlying PD-1-mediated inhibition remain unclear. A better understanding of PD-1 signaling has the potential to identify novel targets for single agent or rationale combination therapy strategies to improve patient responses in cancer. Ligation of PD-1 by PD-L1/L2 results in the attenuation of downstream proliferative pathways, decreases in effector cytokines and cytoskeletal rearrangements. PD-1 exerts its inhibitory function by recruiting tyrosine phosphatases SHP2 and/or SHP1 to tyrosine phosphorylation motifs located on the cytoplasmic tail of PD-1 to dephosphorylate TCR-associated targets. Given the dramatic changes observed in T cells upon PD-1 ligation and the complexity of membrane bound signaling, we believe additional proteins are necessary to mediate PD- 1 inhibitory signals and attenuate the function of unidentified target proteins to influence cancer pathogenesis. My studies using proximity labelling around the cytoplasmic tail of PD-1 upon PD-L1 ligation and subsequent proteomic analysis have identified mechanosensitive cation channel PIEZO1 as the top candidate hit protein. The activation of PIEZO1 through shear stress or lateral membrane stretching plays a role in the development, differentiation and dynamics of various tissues. Recent data show that PIEZO1 can regulate immune cell responses. TCR engagement activates PIEZO1 resulting in calcium influx that contributes to T cell priming, TCR signaling and actin cytoskeletal modulation; all of these processes are targeted by PD-1-mediated inhibition. For these reasons, we hypothesize that PD-1 co-localizes with PIEZO1 to attenuate calcium influx thereby disrupting TCR-mediated signaling, impairing T cell function and attenuating tumor responses. Understanding the consequence of perturbing this potential signaling axis will provide mechanistic insights into PD-1 function and should reveal how PD-1-PIEZO interaction influences cancer pathogenesis. Preliminary data as well as published research suggest PD-1-mediated inhibition is more complex than previously characterized. For this reason, the primary goals of this research are (1) to validate the role of PD-1 in regulating candidate hit protein PIEZO1 and deepen the understanding of PD-1 signaling and (2) to determine how PD-1 and PIEZO regulate anti-tumor immunity. Successful completion of this proposal not only will aid in characterizing PD-1-mediated signaling for identification of potential therapeutic targets in cancer, but also will yield methods that can be applied to the many immunoreceptors for which signaling remains unclear.